Lactobacillus Gasseri KBL697 Strain and Use Thereof

ABSTRACT

A strain of  Lactobacillus gasseri  KBL697 and the use thereof are described. The strain of  Lactobacillus gasseri  KBL697 (Accession No. KCTC 13520BP) attenuates allergic reactions of cells, significantly improves symptoms of atopic dermatitis, and exhibits anti-inflammatory and anti-fungal effects and a therapeutic effect for intestinal diseases such as irritable bowel syndrome and colitis. Thus, the single strain alone can achieve all the purposes of alleviating allergic diseases and inflammatory diseases and improving intestinal health, thereby finding advantageous applications as a probiotic substance. In addition, the strain, based on the anti-fungal activity thereof, can be advantageously utilized in a skin external preparation against various skin diseases caused by fungi, and in a cosmetic composition and a functional patch for alleviating sensitive skin.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/KR2019/006232, which designates the United States and was filed onMay 23, 2019, published in Korean and claims priority under 35 U.S.C. §119 or 365 to Korean Application No. 10-2018-0058568, filed May 23,2018. The entire teachings of the above applications are incorporatedherein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file:

a) File name: 58881002001_SEQUENCELISTING; created Nov. 19, 2020, 3 KBin size.

TECHNICAL FIELD

The present invention relates to a strain of Lactobacillus gasseriKBL697 and the use thereof. More specifically, the present inventionrelates to a health functional food composition having at least oneeffect selected from the group consisting of alleviation of allergicsymptoms, alleviation of inflammatory symptoms, improvement ofintestinal health, and immunoregulation; an anti-fungal composition; anda pharmaceutical composition for the treatment of at least one diseaseselected from the group consisting of allergic diseases, inflammatorydiseases, intestinal diseases, and autoimmune diseases, comprising atleast one selected from the group consisting of a novel probiotic strainof Lactobacillus gasseri KBL697, cultures of said strain, lysates ofsaid strain, and extracts of said strain.

BACKGROUND ART

Probiotics refer to microorganisms and the resulting products therefromhaving antimicrobial activities and enzyme activities to help thebalance of intestinal microorganisms. In addition, probiotics are alsodefined as live bacteria in the form of a single or multiple strain(s)to improve intestinal flora when provided to human or animals in theform of dry cells or fermentation products. Probiotics must inhabit thehuman gut, be non-pathogenic and non-toxic, and survive long enoughuntil they arrive at the intestine. Further, probiotics must maintainviability and activities until they are consumed in the food delivered,be sensitive to antibiotics used to prevent infection, and do not haveantibiotic-resistant plasmids. Also, probiotics must be resistant toacids, enzymes, and bile in the intestinal environment.

These probiotics may include, for example, Bacillus sp. having anexcellent ability to produce digestive enzymes such as amylase,protease, lipase, cellulase, and phosphatase, Lactobacillus sp.producing lactic acid, and photosynthetic bacteria preventing stink byway of using the stink-causing substances (such as ammonia, hydrogensulfide, and amines) remaining in the feces of livestock in metabolicprocess. Recently, probiotics have been reported to have various healthfunction improvement effects including improvement of intestinal health,and thereby spotlighted as major therapeutic substances which canreplace existing compound-based therapeutic agents.

Meanwhile, allergy is a biochemical phenomenon that exhibits a unique,altered response to a foreign substance (antigen, allergen). The foreignsubstance which causes symptoms is called allergen, while the diseasesfrom those symptoms are called allergic diseases. Allergy is apathological process in the living body resulting from theantigen-antibody reaction. In general, there are four types of allergiesdepending on the period to trigger the reaction and the complementinvolvement. Type 1, among those, is anaphylactic type (immediate type)in which target organs are mostly digestive organs, skin and lungs, andthe common symptoms include gastrointestinal allergy, urticaria,atrophodermatitis, allergic rhinitis, and bronchial asthma, etc. Thepathological mechanism of Type 1 is known as follows: when antigenscontact IgE antibodies attached to the surface of mast cells andbasophilic leukocytes, the target cells are activated to secretechemical transmitters such as histamine, leukotriene, and PAF, and thenblood vessels and smooth muscles are contracted. Such mechanism can beoften combined with Type 4 (delayed type). In other words, suchanaphylaxis and allergic reaction can arise due to a variety of changesin the mast cells, etc. The activation of mast cells, which leads todegranulation, is caused by binding of antigen, anti-IgE, lectin, etc.to Fc receptors, stimulation of anaphylatoxin, etc., or other drugs suchas calcium ionophore, compound 48/80, codeine and syntheticadrenocorticotropic hormone.

Mast cells and basophil leukocytes in blood are known as main cells inthe body to cause many allergic diseases such as allergic rhinitis,allergic dermatitis, asthma, food allergy and anaphylactic shock. Thesecells have receptors (FcRI) on their surfaces for IgE which is anantibody causing allergy, and the cells are stimulated by theallergy-causing substances (antigen, allergen) to secrete their ownvarious allergy-causing substances out of the cells (Kim K et al, Eur JPharmacol, 581:191-203, 2008).

Among allergic diseases, atopic dermatitis, as widely known to thepublic, is a chronic recurrent skin disease that affects newborns orchildren and may persist until adulthood. Like asthma or allergicrhinitis, atopic dermatitis is an inflammatory skin disease associatedwith local infiltration of T-lymphocyte which produces IL-4 and IL-5.IL-4, as well known to the public, controls the development of the Thelper 2 (Th2) phenotype, resulting in overproduction of immunoglobulins(Ig) and eosinophilia, and increase of serum IgE levels. 80-90% of thesubjects who were positive to the skin test regarding food and inhalantallergens were found to have atopic dermatitis.

There are different treatments for treating or preventing allergicdiseases and atopic dermatitis, but no effective treatment has beenfound yet. Some drug-based treatments are known, but even a short termadministration of the drug for the treatment would develop a toleranceand a long-term administration may cause serious side effects, and thussuch drug-based treatments of allergic diseases and atopic dermatitishave been avoided recently. Under the circumstances, without treatmenthaving any absolute, obvious effect, irritating symptoms such as itchingand redness of skin in addition to allergy often fail to improve.

Meanwhile, irritable bowel syndrome (IBS) is a symptom characterized byabdominal pain, and/or irritations associated with changed intestinalmovement or bowel habits, such symptoms cannot be explained withanatomic or biochemical abnormality. Common symptoms of IBS also includeurinary urgency, bloating and feeling of incomplete intestinal movement.Accordingly, IBS can be classified as functional gastrointestinaldisorders comprising conditions such as functional bloating, non-cardiacchest pain, non-ulcerative dyspepsia, and chronic constipation ordiarrhea. In particular, in the case of IBS, since the related symptomsaffect both well-being and normal function aspects of patients, thedisease has a huge impact on morbidity and quality of life, beyondabdominal pain and discomfort.

Inflammatory bowel disease (IBD) is a condition in which abnormalchronic inflammation in the intestine repeats improvement andrecurrence, comprising all intestinal inflammatory diseases, such asCrohn's disease, ulcerative colitis, or Behcet's disease, but notlimited thereto. Many researches have been conducted in the field ofdrug development to treat IBS and IBD. In this regard, variousantidepressants are commonly used, even though the efficacy thereof inclinical trials is moderate and the clinical utility thereof is limiteddue to significant side effects. Serotonergic medications have also beenproved to have efficacy against overall IBS symptoms. However, theapplication of these medications has been restricted in various ways dueto recent several safety problems. Accordingly, there is increasinginterest in developing a new therapeutic agent for IBS.

WO 96/29083 and EP 554418 disclose two types of Lactobacillus strainswhich form colonies in bowel, i.e., Lactobacillus plantarum 299v (DSM6595) and Lactobacillus casei ssp. rhamnosus 271 (DSM 6594), etc. EP415941 discloses a method for preparing a nutrient composition,comprising treating oat gruel with enzymes before mixing it withlactobacilli. U.S. Pat. No. 7,195,906 discloses a strain ofBifidobacterium isolated from resected and washed human gastrointestinaltract for the treatment of inflammatory diseases, especiallygastrointestinal inflammatory activity such as IBD and IBS.

However, no strain having excellent effects on improving intestinalhealth, for example, treatment of IBD and IBS has been found yet, and inorder to find strains having such effects, many research institutionshave been working on.

Under the circumstances, the present inventors devoted themselves tostudies of probiotics to find a way to replace drug-based treatments forallergic diseases, including atopic dermatitis, which have nosatisfactory treatments. And therefore, the present invention wascompleted by confirming that a novel strain of Lactobacillus gasserishowed excellent therapeutic effects on allergic diseases such as atopicdermatitis, and further confirming that said strain also showed superioreffects on anti-fungal activity, intestinal health, immunoregulation,and inhibition of inflammation.

SUMMARY

The purpose of the present invention is to provide a novel strainshowing excellent effects on alleviation of allergic symptoms such asatopic dermatitis, alleviation of inflammatory symptoms, anti-fungalactivity, improvement of intestinal health, and immunoregulation, and avariety of uses thereof.

In order to achieve the purpose, the present invention providesLactobacillus gasseri KBL697 strain (Accession No. KCTC 13520BP).

Also, the present invention provides a food composition or food additivecomposition comprising at least one selected from the group consistingof said strain, cultures of said strain, lysates of said strain, andextracts of said strain.

The present invention provides a feed composition or feed additivecomposition comprising at least one selected from the group consistingof said strain, cultures of said strain, lysates of said strain, andextracts of said strain.

The present invention also provides an anti-fungal composition, such asan anti-dandruff composition, comprising at least one selected from thegroup consisting of said strain, cultures of said strain, lysates ofsaid strain, and extracts of said strain.

The present invention also provides a pharmaceutical composition for thetreatment of allergic diseases such as atopic dermatitis, inflammatorydiseases, intestinal diseases and/or autoimmune diseases, comprising atleast one selected from the group consisting of said strain, cultures ofsaid strain, lysates of said strain, and extracts of said strain.

The present invention also provides a method for treating allergicdiseases including atopic dermatitis, inflammatory diseases, intestinaldiseases and/or autoimmune diseases, comprising administering at leastone selected from the group consisting of said strain, cultures of saidstrain, lysates of said strain, and extracts of said strain to a subjectin need thereof.

The present invention also provides a composition comprising at leastone selected from the group consisting of said strain, cultures of saidstrain, lysates of said strain, and extracts of said strain, for the useof preventing or treating allergic diseases including atopic dermatitis,inflammatory diseases, intestinal diseases and/or autoimmune diseases.

The present invention also provides the use of a composition forpreparing a preventive or therapeutic drug for allergic diseasesincluding atopic dermatitis, inflammatory diseases, intestinal diseasesand/or autoimmune diseases, comprising at least one selected from thegroup consisting of said strain, cultures of said strain, lysates ofsaid strain, and extracts of said strain.

The present invention also provides a cosmetic composition comprising atleast one selected from the group consisting of said strain, cultures ofsaid strain, lysates of said strain, and extracts of said strain.

The present invention also provides a cosmetic patch or a medical patchcomprising at least one selected from the group consisting of saidstrain, cultures of said strain, lysates of said strain, and extracts ofsaid strain.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the result confirming the inhibitory effect of IL-4expression by various Lactobacillus strains including Lactobacillusgasseri KBL697 strain, after inducing allergic reaction in EL4 celllines.

FIG. 2 illustrates the result confirming the inhibitory effect of IL-5expression by various Lactobacillus strains including Lactobacillusgasseri KBL697 strain, after inducing allergic reaction in EL4 celllines.

FIG. 3 illustrates the result confirming an inhibitory effect ofhistamine secretion by the treatment of various Lactobacillus gasseristrains, and an antihistaminic agent ketotifen, after inducing histamineproduction by antigen-antibody reaction in RBL 2H3 cell lines.

FIG. 4 illustrates the result of observation of the remarkable effect ofincreasing the amount of anti-inflammatory, immunoregulatory cytokineIL-10 secretion by the treatment of the KBL697 strain, when treatingwith Lactobacillus gasseri strains after inducing inflammatory reactionin RAW 264.7 cell lines.

FIG. 5 illustrates the result confirming the remarkable immunoregulatoryand anti-inflammatory effect by the treatment of the KBL697 strain withan IL-10/TNF-α value, when treating with Lactobacillus gasseri strainsafter inducing inflammatory reaction in RAW 264.7 cell lines.

FIG. 6 illustrates the result confirming the remarkable immunoregulatoryand anti-inflammatory effect by the treatment of the KBL697 strain withan IL-10/IL-6 value, when treating with the Lactobacillus gasseristrains after inducing inflammatory responses in RAW 264.7 cell lines.

FIG. 7 illustrates the result of a spot assay confirming the anti-fungalactivity of Lactobacillus gasseri KBL697 strain.

FIG. 8 illustrates the result confirming the dermatitis score reducingeffect by the oral administration of Lactobacillus gasseri KBL697 strainto mouse models that atopic dermatitis was induced.

FIG. 9 illustrates the result confirming the itching-alleviating effectby the oral administration of Lactobacillus gasseri KBL697 strain tomouse models that atopic dermatitis was induced.

FIG. 10 illustrates the result confirming the ear thickness loweringeffects by the oral administration of Lactobacillus gasseri KBL697strain to mouse models that atopic dermatitis was induced.

FIG. 11 illustrates the result confirming the skin thickness loweringeffect by the oral administration of Lactobacillus gasseri KBL697 strainto mouse models that atopic dermatitis was induced.

FIG. 12 illustrates the result confirming the IgE concentration-in-bloodlowering effect by the oral administration of Lactobacillus gasseriKBL697 strain to mouse models that atopic dermatitis was induced.

FIG. 13 illustrates the result of a TEER assay confirming the effect ofstrengthening tight junctions by Lactobacillus gasseri KBL697 strain.

FIG. 14 illustrates the result of observation of the body weightrecovering effect by the oral administration of Lactobacillus gasseriKBL697 strain at 1×10⁹ CFU to mouse models that colitis was induced.

FIG. 15 illustrates the result of observation of the large intestinelength recovering effect by the oral administration of Lactobacillusgasseri KBL697 strain to mouse models that colitis was induced.

FIGS. 16A-16G illustrate the results of observation of the alleviatingeffect of PASI (Psoriasis Area and Severity Index) and edema symptoms byLactobacillus gasseri KBL697 strain in mouse models that psoriasis wasinduced.

FIGS. 17A-17D illustrate the determination results of the effect oflowering inflammatory cytokines (TNF-α, IFN-γ, IL-17) by Lactobacillusgasseri KBL697 strain in mouse models that psoriasis was induced.

FIGS. 18A-18C illustrate the results of comparing the effect ofrecovering body weight and the length of the large intestine byLactobacillus gasseri KBL697 strain and infliximab in mouse models thatcolitis was induced.

BEST MODE

Unless defined otherwise, all of the technical, scientific terms used inthe present specification mean the same as understood by a person havingordinary skills in the art (“those skilled in the art”). In general, thenomenclature used in the present specification is well known in the artand commonly used.

The present invention has found an anti-allergic effect ofmicroorganisms derived from the human body, and selected Lactobacillusgasseri KBL697 strain (Accession No. KCTC 13520BP) having excellentallergy inhibitory effects. Analysis of 16S rDNA of said straindemonstrates that said strain is a novel strain which has never beenknown to the public.

According to one embodiment of the present invention, the presentinvention relates to a novel probiotic strain of Lactobacillus gasseriKBL697 (Accession No. KCTC 13520BP), and said strain is characterized bycomprising 16S rDNA sequence of SEQ ID NO: 1.

16S rDNA sequence of a strain of Lactobacillusgasseri KBL697 (Accession No. KCTC 13520BP) <SEQ ID NO: 1>GGCAAGTGGGCGGCGTGCTATACATGCAGTCGAGCGAGCTTGCCTAGATGAATTTGGTGCTTGCACCAAATGAAACTAGATACAAGCGAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAACAGATGCTAATACCGGATAACAACACTAGACGCATGTCTAGAGTTTAAAAGATGGTTCTGCTATCACTCTTGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGCAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATTACTTAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTCGGCTCAACCGGAGAATTGCATCAGAAACTGTTGAACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTGCAAACCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGAAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGAAGCGAACCTGCGAAGGCAAGCGGATCTCTGAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTCTGTAACACCCAAAGCCGGTGGGATAACCTTTATAGGAGTCAGCCGTCTAAGTAGACAGATGTTA

Then, the present invention conducted experiments regarding the efficacyof said strain, and thereby verified that said strain has an excellentinhibitory effect on allergies such as atopic dermatitis, alleviates theinflammatory reaction, and has an anti-fungal activity, immunoregulatoryproperties and therapeutic effects on intestinal diseases. Further, theinventors confirmed that said effects could be provided not only in thecondition of living bacteria but also under the low temperaturesterilization or the high temperature sterilization.

Accordingly, in another embodiment of the present invention, the presentinvention relates to a food composition or food additive compositioncomprising at least one selected from the group consisting ofLactobacillus gasseri KBL697 strain (Accession No. KCTC 13520BP),cellular components of said strain, cultures of said strain, lysates ofsaid strain, and extracts of said strain.

The composition can be characterized in that it is a health functionalfood composition having at least one effect selected from the groupconsisting of alleviation of allergic symptoms such as atopicdermatitis, alleviation of inflammatory symptoms, improvement ofintestinal health, and immunoregulation.

Said food composition or food additive composition can be readilyutilized as the food effective for alleviation of allergic symptoms suchas atopic dermatitis, alleviation of inflammatory symptoms, improvementof intestinal health and/or immunoregulation, and for the preventionthereof, for example, as main ingredients or minor ingredients of food,food additives, health functional food composition or functionalbeverages, but not limited thereto.

The term “food composition” refers to a natural or artificial productcomprising at least one nutrient, and more preferably, refers to aproduct which became edible through certain processing, usuallyencompassing all of food, food additives, health functional food andfunctional beverages.

The food that may comprise the said food composition according to thepresent invention as an additive may include, for example, differenttypes of food, beverages, chewing gum, tea, vitamin complex, orfunctional food. In addition, the food of the present invention includesspecial nutritional food (e.g., modified milk, infant/baby food),processed meat products, fish meat products, tofu, muk, noodles (e.g.,ramen, Asian noodles), bakery products, health supplement food,seasoning products (e.g., soy sauce, soybean paste, red pepper paste,mixed paste), sauces, confectionery (e.g., snack foods), candies,chocolates, chewing gums, ice-creams, milk products (e.g., fermentedmilk, cheese), other processed food, Kim-chi, salted food (e.g.,different types of Kim-chi, pickled food), beverages (e.g., fruit juice,vegetable juice, soy milk, fermented beverages), and natural seasonings(e.g., broth powder for ramen), but not limited thereto. Said food,beverages or food additives can be prepared in conventional manners.

The term “health functional food” is a group of food to which value isadded so as for the function thereof to be exerted and expressed for thepredetermined purpose by using physical, biochemical or bioengineeringtechniques thereto, or a processed food designed so as for the in-vivoadjustment functions of the relevant food composition such as rhythmadjustment in prophylaxis, prevention of disease and recovery fromdisease to be sufficiently expressed. Such functional food may comprisefood supplement additives which are food-scientifically acceptable, andmay additionally comprise suitable carriers, excipients and diluents,which are commonly used in the manufacturing thereof.

The term “functional beverages”, as used in the present invention,collectively refer to the drink products to relieve thirst or to enjoythe taste. There is no particular limitation thereto, except that, asessential ingredients of the indicated ratio, a composition foralleviation of allergic symptoms such as atopic dermatitis, alleviationof inflammatory symptoms, improvement of intestinal health and/orimmunoregulation and the prevention thereof should be comprised in thebeverages, and various flavoring agents or natural carbohydrates may becontained therein as additional ingredients like in common beverages.

In addition to the above, the food comprising the food composition orthe food additive composition according to the present invention maycontain various nutrients, vitamins, minerals (electrolyte), flavoringagents such as synthetic flavoring agents and natural flavoring agents,coloring agents and fillers (cheese, chocolate, etc.), pectic acid andsalts thereof, alginic acid and salts thereof, organic acids, protectivecolloidal thickening agents, pH controlling agents, stabilizing agents,preservatives, glycerin, alcohol, carbonizing agents as used incarbonated beverages and the like, and each of the above ingredients maybe used alone or in combination with each other.

In the food comprising the food composition according to the presentinvention, the composition of the present invention may be comprised inan amount of 0.001% by weight to 100% by weight, and preferably 1% byweight to 99% by weight, based on the total weight of the food; in thecase of beverages, it may be comprised at an amount of 0.001 g to 10 g,and preferably 0.01 g to 1 g, based on 100 ml. For long-term intake forthe purpose of health and hygiene or for the purpose of health control,however, the amount may be below the above-mentioned range; and sincethe effective ingredients have no problem in terms of safety profile,they can be used at an amount above the range and they are not limitedto the amount range mentioned above.

The food composition according to the present invention may compriseLactobacillus gasseri KBL697 strain alone or in combination with theacceptable carrier, or may be prepared in the form of the compositionsuitable for consumption by human or animals. That is, the compositionmay be added to the food which comprises no probiotic bacteria or acouple of probiotic bacteria. For example, the microorganisms which canbe used in combination with the strain according to the presentinvention in preparing the food of the present invention should besuitable for the consumption by human or animals, and have probioticactivities to inhibit pathogenic, harmful bacteria or to improve thebalance of microorganisms in the mammalian intestinal tract, uponintake, but not limited thereto. Such probiotic microorganisms mayinclude, for example, yeast such as Saccharomyces, Candida, Pichia orTorulopsis, fungi such as Aspergillus, Rhizopus, Mucor, or Penicillium,and bacteria belonging to the genus of Lactobacillus, Bifidobacterium,Leuconostoc, Lactococcus, Bacillus, Streptococcus, Propionibacterium,Enterococcus, or Pediococcus. Suitable probiotic microorganismsspecifically may include, for example, Saccharomyces cerevisiae,Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Enterococcus faecium, Enterococcus faecalis, Lactobacillusacidophilus, Lactobacillus alimentarius, Lactobacillus casei,Lactobacillus curvatus, Lactobacillus delbruckii, Lactobacillusjohnsonii, Lactobacillus farciminus, Lactobacillus gasseri,Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillusreuteri, Lactobacillus sakei, Lactococcus lactis, or Pediococcusacidilactici. Preferably, the food composition according to the presentinvention may further comprise a probiotic microorganism mixture havingexcellent probiotic activities and superior activities of anti-allergy,anti-inflammation, immunoregulation and/or improvement of intestinalhealth to further enhance the effects thereof. The carriers that can beincluded in the food composition of the present invention may include,for example, extenders, high fiber additives, encapsulating agents, andlipids, which are widely well known in the art. Lactobacillus gasseriKBL697 strain in the present invention may be in the lyophilized orencapsulated form or in the form of culture suspensions or dry powders.

The composition of the present invention can also be provided in theform of a feed additive comprising said strain or a feed comprising thesame.

The feed additive of the present invention may be in the form of dry orliquid formulation, and further comprise other non-pathogenicmicroorganisms in addition to the said Lactobacillus gasseri KBL697strain. The microorganisms that can be added to the feed additive mayinclude, for example, Bacillus subtilis that can produce protease,lipase and sugar-converting enzymes, Lactobacillus strain having aphysiological activity and degradability of organic compounds underanaerobic conditions such as in the stomach of cow, filamentous fungisuch as Aspergillus oryzae showing effects on increasing weight ofanimals, milk yield, and digestibility of the feed (Slyter, L. L. J.Animal Sci., 1976, 43. 910-926) and yeast such as Saccharomycescerevisiae (Johnson, D. E et al. J. Anim. Sci., 1983, 56, 735-739;Williams, P. E. V. et al, 1990, 211).

The feed additive of the present invention may additionally comprise atleast one enzyme agent in addition to said strain of Lactobacillusgasseri KBL697. The additional enzyme agents can be in a dry or liquidform, and may include, for example, steatolytic enzymes such as lipase,phytase to produce phosphate and inositol phosphate by degrading phyticacid, amylase, i.e., an enzyme to hydrolyze α-1,4-glycoside bondincluded in, for example, starch and glycogen, phosphatase, i.e., anenzyme to hydrolyze organic phosphoric acid ester,carboxymethylcellulase to degrade cellulose, xylase to degrade xylose,maltase to hydrolyze maltose into two glucose molecules, and sugarproducing enzymes such as invertase to produce glucose-fructose mixtureby hydrolyzing saccharose.

In the use of Lactobacillus gasseri KBL697 strain of the presentinvention as feed additives, the raw ingredients for the feed, such aspeanuts, peas, beets, pulp, grain by-products, animal guts powder andfish meal powder, including various grains and soybean protein, can beused. They may be processed or not, and can be used without limitation.The processing may include, but not limited thereto, such a process thatthe raw ingredients of the feed are charged and can be compressed underpressure against a given outlet, and for proteins, extrusion by whichproteins are degenerated to increase availability may be preferablyused. Extrusion denatures proteins through thermal treatment to destroyantienzyme factors, which is advantageous. Further, for soybeanproteins, the digestibility thereof can be improved through extrusion toinactivate anti-nutrients such as a trypsin inhibitor, one of inhibitorsof protease that are present in soybeans. Further, extrusion can promoteimprovement of digestibility by protease, enhancing the nutritionalvalue of soybean proteins.

According to another embodiment of the present invention, the presentinvention relates to an anti-fungal composition comprising at least oneselected from the group consisting of said strain, cultures of saidstrain, lysates of said strain, and extracts of said strain.

The composition can be characterized by showing an anti-fungal activityon the one selected from the group consisting of Malassezia furfur,Malassezia globosa and Malassezia restricta, but not limited thereto.

The composition can be a composition for preventing, alleviating ortreating seborrheic dermatitis or dandruff, and said seborrheicdermatitis can be on scalp.

Further, said composition can be a composition for preventing,alleviating or treating urticaria, rash, tinea corporis, tinea cruris,or tinea pedis, due to mycotic infection.

Said strain, and cultures of said strain, lysates of said strain, andextracts of said strain can be comprised in an amount of 0.1% by weightto 50% by weight, based on the total weight of the composition.

Said anti-fungal composition may be a pharmaceutical composition, acosmetic composition or a health food composition, and it can also be askin external preparation.

Said cosmetic composition can be provided in all dosage forms which aresuitable for topical application, for example, in the form of liquid,oil-in-water type emulsion, water-in-oil type emulsion, suspension,solid, gel, powder, paste, foam or aerosol. Said compositions of theabove dosage forms can be prepared in conventional methods used in theart.

In addition to the above ingredients, said composition may compriseother ingredients at an amount which does not harm the main effect,preferably, at an amount to provide a synergistic effect on the maineffect. The composition according to the present invention may comprisea substance selected from the group consisting of vitamins,polypeptides, polysaccharides, and sphingolipid. Further, the cosmeticcomposition of the present invention may comprise a moisturizer, anemollient agent, a surfactant, a UV absorbent, a preservative, asterilizer, an antioxidant, a pH adjusting agent, organic and inorganicpigments, flavoring agent, a cooling agent or an antiperspirant agent.The combination percentage of said ingredients can be selected by thoseskilled in the art within the range not to hinder the purpose and effectof the present invention, and can be in a range from 0.01% by weight to5% by weight, and specifically from 0.01% by weight to 3% by weight,based on the total weight of the composition.

According to the above embodiment, the anti-fungal composition of thepresent invention can be a skin external preparation such as cream,ointment, shampoo, or treatment.

According to another embodiment of the present invention, the presentinvention relates to a pharmaceutical composition for the treatment orprevention of allergic diseases including atopic dermatitis,inflammatory diseases, intestinal diseases and/or autoimmune diseases,comprising at least one selected from the group consisting of cellularcomponent of Lactobacillus gasseri KBL697 strain, cultures of saidstrain, lysates of said strain, and extracts of said strain.

The pharmaceutical composition of the present invention can be providedin a form of cellular component of live bacteria, dry strain, culturesof said strain, lysates of said strain, or a composition in combinationwith a pharmaceutically acceptable carrier or media. The carriers ormedia that can be used herein may include solvent, a dispersant, acoating, an enhancer, a controlled-release formulation (i.e.,sustained-release formulation), or at least one inert excipientincluding starch, polyol, granules, microtine cellulose,microcrystalline cellulose such as Celphere, Celphere beads, diluent,lubricant, binder, disintegrant. The tablet of the above composition maybe, if desired, coated by a standard aqueous or non-aqueous technique.The examples of the pharmaceutically acceptable carrier and theexcipient for the use as the pharmaceutically acceptable inert carrier,and said additional ingredients may include, for example, a binder, afiller, a disintegrant, a lubricant, an antimicrobial agent and acoating agent, but not limited thereto.

Further, the pharmaceutical composition of the present invention can beused as an external preparation comprising a dosage form selected fromthe group consisting of ointments, creams, pastes, liquids and solutionsfor cutaneous application, glycerogelatins, liniments, powders forcutaneous application, aerosols, and plasters.

In the present invention, said allergic diseases refer to the conditionsassociated with IL-4 or IL-5 expressions, and may include, for example,eczema, allergic asthma, atopic dermatitis, allergic rhinitis, allergicconjunctivitis, urticaria, or anaphylaxis. Preferably, the presentinvention may be characterized in that the diseases are selected fromthe group consisting of infant eczema, allergic asthma, allergicrhinitis, atopic dermatitis, allergic conjunctivitis, and food allergy,but not limited thereto.

The present invention may be characterized in that said intestinaldiseases are selected from the group consisting of abdominal bloating,abdominal discomfort, infectious diarrhea caused by pathogenicmicroorganisms, gastrocolitis, inflammatory bowel diseases, neurogenicalintestinitis syndrome, irritable bowel syndrome, overgrowth of smallintestinal microorganisms and intestinal feeding diarrhea, and thediseases also include those caused by damage of barrier function ofintestine.

The inflammatory bowel disease (IBD) may include Crohn's disease, theintestinal lesion accompanying with Behcet's disease, ulcerativecolitis, hemorrhagic rectal ulcer, and pouchitis, and refers to a groupof diseases including Crohn's disease and ulcerative colitis. Ulcerativecolitis only affects the large intestine. Inflammation and ulcer ofulcerative colitis are limited to the two innermost layers, mucosa andsubmucosa out of four layers of the large intestine. Inflammation andulcer of Crohn's disease can be spread throughout all layers of theintestinal wall in both small and large intestines.

Meanwhile, the irritable bowel syndrome is a chronic conditionaccompanying not only persistently recurrent abdominal discomfort andpain such as abdominal bloating, but also changes in bowel habit such asdiarrhea and constipation. The symptoms may be exacerbated bypsychological factors or stressful social circumstances.

In the present invention, the inflammatory diseases collectively referto conditions having inflammation as a main lesion, and may include, forexample, edema, conjunctivitis, periodontitis, rhinitis, otitis media,chronic sinusitis, pharyngolaryngitis, tonsillitis, bronchitis,pneumonia, gastric ulcer, gastritis, colitis, gout, eczema, acne, atopicdermatitis, contact dermatitis, seborrheic dermatitis, ankylosingspondylitis, rheumatic fever, fibromyalgia, osteoarthritis, psoriaticarthritis, rheumatoid arthritis, peri-arthritis of the shoulder,tendinitis, tenosynovitis myositis, hepatitis, cystitis, nephritis,Sjogren's syndrome, myasthenia gravis, sepsis, vasculitis, bursitis,temporal arteritis, and multiple sclerosis, but not limited thereto.

In the present invention, autoimmune diseases and the symptoms that canbe alleviated by the immunoregulatory effect may include, for example,rheumatoid arthritis, lupus, systemic scleroderma, atopic dermatitis,psoriasis, psoriatic arthritis, asthma, Guilian-Barre syndrome,myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis,autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto'sthyroiditis, multiple sclerosis, temporal arteritis, juvenile diabetes,alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolyticanemia, Wegener's granulomatosis, Sjogren's syndrome, Addison's disease,Crohn's disease, and Behcet's disease, but not limited thereto.

The present invention may be characterized in that the effects resultingfrom the strain of the present invention for immunoregulation, or foralleviating, treating or preventing autoimmune diseases or inflammatorydiseases can be induced by at least one mechanism selected from increaseand decrease of cytokines associated with inhibition of inflammation andimmunoregulation, specifically decrease of TNF-α, IFN-γ, and IL-17secretion and increase of IL-10 secretion.

TNF-α (tumor necrosis factor-α) is a 17 kDa peptide produced bymacrophages and various other cells activated during the host immunereaction to bacterial infections and oncologic diseases. This cytokineis also known as an important mediator of immune and inflammatoryreactions, and also known as a proinflammatory cytokine which plays animportant role in autoimmune diseases and inflammatory diseases such asrheumatoid arthritis (RA), psoriatic arthritis, Crohn's disease,psoriasis, and ankylosing spondylitis (AS).

On the other hand, CD4⁺ T cells can be classified into subtypes such asTh1 (T helper type 1), Th2 (T helper type 2), CD4⁺ CD25⁺immunoregulatory T cell and Th17 cells (T helper 17 cell) depending onthe expression of specific cytokines and transcription factors. Amongthose, IL-17 produced by Th17 cells is known as mostly involved inautoimmune diseases, allergic reactions, or host defense againstbacterial or fungal infection.

Further, IL-10 is a peptide of 35-40 kDa produced by helper T cell, Bcell, monocyte, and other cells, and has immunosuppressive andanti-inflammatory properties, for example, inhibition of production ofcytokines including TNF-α, and IFN-γ.

The term ‘treating’, unless mentioned otherwise, refers to reversing oralleviating the diseases or conditions used with said term or one ormore symptoms thereof, inhibiting the progression of the same orpreventing the same. The term ‘treatment’ as used in the presentinvention refers to an act of ‘treating’ as defined above. Accordingly,treatment or therapeutic regimen of a disease in mammals may include oneor more of the following:

(1) Inhibit the growth of the disease, that is, inhibit its development

(2) Prevent the spread of the disease

(3) Alleviate the disease

(4) Prevent recurrence of the disease, and

(5) Palliate the symptoms of the disease

A composition of the present invention for preventing or treatingallergic diseases such as atopic dermatitis, inflammatory diseases,intestinal diseases and/or autoimmune diseases may comprise apharmaceutically effective amount of Lactobacillus gasseri KBL697 strainalone or in combination of with at least one of pharmaceuticallyacceptable carriers, excipients or diluents.

In the present invention, the term “effective amount” means an amountthat is high enough to provide a desired effect but is low enough toprevent serious side effects under medical judgment. The amount ofmicroorganisms administered to the body by the composition of thepresent invention can be appropriately adjusted in consideration of theadministration route and the administration target.

The composition of the present invention can be administered to asubject once or more per day. Unit dosage means physically discreteunits suitable for unit administration to human subjects and othermammals, and each unit comprises a suitable pharmaceutical carrier and apredetermined amount of Lactobacillus gasseri KBL697 strain of thepresent invention to provide a therapeutic effect. The dosage unit fororal administration to an adult patient preferably contains 0.001 g ormore of the microorganism of the present invention, and the oral dosageof the composition of the present invention is from 0.001 g to 10 g, andpreferably from 0.01 g to 5 g per dose. The pharmaceutically effectiveamount of the microorganism of the present invention is from 0.01 g to10 g/day. However, the dosage varies depending on the severity of thepatient's disease and the microorganisms and auxiliary effectiveingredients used together. In addition, the total daily dosage can bedivided into several times and continuously administered as needed.Accordingly, the above dosage ranges do not limit the scope of thepresent invention in any way.

Further, the term “pharmaceutically acceptable” as used above refers toa composition that is physiologically acceptable and does not cause anallergic reaction such as gastrointestinal disorder, or dizziness, orsimilar reaction when administered to a human.

A composition of the present invention can be formulated using methodsknown in the art so that rapid, sustained or delayed release of theactive ingredients, after administered to a mammal, can be provided. Thedosage forms may be powders, granules, tablets, emulsions, syrups,aerosols, soft or hard gelatin capsules, sterile injection solutions, orsterile powders. Further, the composition of the present invention forpreventing or treating allergic diseases such as atopic dermatitis,inflammatory diseases, intestinal diseases and/or autoimmune diseasescan be administered via several routes, including oral, transdermal,subcutaneous, intravenous or intramuscular administration. The dosage ofthe active ingredients can be appropriately selected depending onvarious factors such as the route of administration, the patient's age,sex, weight, and the severity of the patient. The composition of thepresent invention for treating allergic diseases such as atopicdermatitis, inflammatory diseases, intestinal diseases and/or autoimmunediseases can be administered in combination with a known compound havingthe effect of preventing, alleviating or treating the relevant symptoms.

The pharmaceutical composition of the present invention, in particular,can be provided in an oral unit dosage form of an enteric coatedformulation. The term “enteric coating”, as used herein, comprises anyknown pharmaceutically acceptable coating which can remain in thestomach without degrading by the gastric acid and can sufficientlydisintegrate in the intestinal tract to release active ingredientstherein. The “enteric coating” of the present invention refers to acoating that can be maintained for 2 hours or more when an artificialgastric juice such as an HCl solution of pH 1 is contacted thereto at36° C. to 38° C., and subsequently can degrade, preferably in anartificial intestinal juice such as a KH₂PO₄ buffer solution of pH 6.8in 30 minutes.

The enteric coating of the present invention is coated on one core in anamount of from about 16 mg to 30 mg, preferably from 16 mg to 20 mg or25 mg or less. When the thickness of the enteric coating of the presentinvention is 5 μm to 100 μm, and preferably 20 μm to 80 μm, satisfactoryresults can be obtained as an enteric coating. The material of theenteric coating can be suitably selected from known polymeric materials.Suitable polymeric materials are listed in a number of known documents(L. Lachman et al., The Theory and Practice of Industrial Pharmacy,3^(rd) ed., 1986, pp. 365˜373; H. Sucker et al., PharmazeutischeTechnologie, Thieme, 1991, pp. 355-359; Hagers Handbuchderpharmazeutischen Praxis, 4^(th) ed., Vol. 7, pp. 739˜742, and 766˜778,(SpringerVerlag, 1971); and Remington's Pharmaceutical Sciences, 13^(th)ed., pp. 1689˜1691 (Mack Publ., Co., 1970)), and cellulose esterderivatives, cellulose ethers, a copolymer of acrylic resin andmethylacrylate, and a copolymer of maleic acid and phthalic acidderivatives can be included therein.

The enteric coating of the present invention can be prepared using aconventional enteric coating method in which an enteric coating solutionis sprayed onto a core. Suitable solvents used for the enteric coatingprocess are alcohols such as ethanol, ketones such as acetone,halogenated hydrocarbon solvents such as dichloromethane (CH₂Cl₂), andmixed solvents of these solvents. A softener such as di-n-butylphthalate or triacetin is added to the coating solution in a ratio of1:about 0.05 to about 0.3 (coating material:softener). It is appropriateto carry out the spraying process continuously, and it is possible toadjust the spraying amount in consideration of the conditions ofcoating. The spraying pressure can be variously adjusted, andsatisfactory results can be obtained generally with a spraying pressureof about 1 bar to about 1.5 bar.

Meanwhile, the pharmaceutical composition of the present invention canbe administered in combination with conventional drugs which are knownto have preventing or treating effect on allergic diseases, inflammatorydiseases, intestinal diseases and/or autoimmune diseases. For example,the drugs that can be administered in combination with thepharmaceutical composition of the present invention may include antibodymedications used for treating inflammatory diseases, intestinal diseasesor autoimmune diseases such as infliximab, adalimumab, golimumab,abciximab, alemtuzumab, atlizumab, basiliximab, belimumab, bevacizumab,ipilimumab, brentuximab vecotin, canakinumab, capromab pendetide,catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab,eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etanercept,etaracizumab, fontolizumab, gemtuzumab ozogamicin, girentuximab,ibritumomab tiuxetan, igovomab, imciromab, ipilimumab, labetuzumab,mepolizumab, motavizumab, natalizumab, nimotuzumab, nofetumomabmerpentan, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab,pemtumomab, pertuzumab, ranibizumab, raxibacumab, rituximab,rovelizumab, ruplizumab, tacatuzumab tetraxetan, tefibazumab,tocilizumab, tositumomab, trastuzumab, ustekinumab, secukinumab,vedolizumab, visilizumab, votumumab, zalutumumab and zanolimumab, butnot limited thereto.

In addition, the pharmaceutical composition of the present invention canbe used in combination with antihistamines, steroids, salicylic acid,urea, tacrolimus, cyclophosphorine, phototherapy, and the like, for thetreatment of eczema and allergic diseases such as atopic dermatitis, andcan be used in combination with sulfasalazine, azathioprine,mercaptopurine, methotrexate, cyclosporine, TNF blocking agents(adalimumab, etanercept, etc.), or integrin antibodies (vedolizumab,natalizumab, etc.), for the treatment of intestinal diseases such asinflammatory bowel diseases.

In another embodiment of the present invention, the present inventionprovides the use of said strain or said composition for preventing ortreating allergic diseases such as atopic dermatitis, inflammatorydiseases, intestinal diseases and/or autoimmune diseases, and the use ofsaid strain or said composition for preparing a therapeutic agent forthe above diseases.

Specifically, the present invention relates to a composition comprisingat least one selected from the group consisting of said strain, culturesof said strain, lysates of said strain, and extracts of said strain, forthe use of preventing or treating allergic diseases including atopicdermatitis, inflammatory diseases, intestinal diseases such as colitis,and/or autoimmune diseases such as psoriasis.

The present invention also relates to the use of a composition forpreparing a preventive or therapeutic drug for allergic diseasesincluding atopic dermatitis, inflammatory diseases, intestinal diseasessuch as colitis, and/or autoimmune diseases such as psoriasis,comprising at least one selected from the group consisting of saidstrain, cultures of said strain, lysates of said strain, and extracts ofsaid strain.

The term ‘prevention’, as used herein, is associated with averting,delaying, impeding or hindering diseases to reduce the same.

The term ‘treatment’, as used herein, is associated with caring for asubject suffering from diseases in order to ameliorate, cure or reducethe symptoms of the diseases or to reduce or stop the progression of thediseases.

In another embodiment of the present invention, the present inventionprovides a method for preventing or treating allergic diseases includingatopic dermatitis, inflammatory diseases, intestinal diseases and/orautoimmune diseases, comprising administering a pharmaceuticallyeffective amount of the strain or said composition to a subject in needof prevention or treatment of said diseases, or in need of alleviationof the intestinal health, allergic reactions, inflammatory reactions orautoimmunity reactions.

Specifically, the present invention provides a method for treatingallergic diseases including atopic dermatitis, inflammatory diseases,intestinal diseases such as colitis, and/or autoimmune diseases such aspsoriasis, comprising administering a therapeutically effective amountof at least one selected from the group consisting of said strain,cultures of said strain, lysates of said strain, and extracts of saidstrain to a subject in need thereof.

Since the pharmaceutical composition used for the method for preventingor treating said diseases, and the administration method thereof havebeen described above, the overlapping contents between the compositionand the method will be omitted herein to avoid excessive complexity ofthe present specification.

Meanwhile, the said subject to which the composition for preventing ortreating said diseases can be administered includes all animalsincluding human. For example, the subject may be an animal such as dog,cat, or mouse.

In another embodiment of the present invention, the present inventionrelates to a cosmetic composition comprising a pharmaceuticallyeffective amount of at least one selected from the group consisting ofthe cellular component of Lactobacillus gasseri KBL697 strain, culturesof said strain, lysates of said strain, and extracts of said strain.

The cosmetic composition may be characterized by its function ofalleviating at least one sensitive skin condition selected from thegroup consisting of cutaneous allergy, skin urticaria, atopicdermatitis, psoriasis, mycotic infections and eczema, but not limitedthereto.

The term ‘cosmetic composition’, as used herein, refers to a compositioncomprising at least one selected from the group consisting of thecellular component of Lactobacillus gasseri KBL697 strain, cultures ofsaid strain, lysates of said strain, and extracts of said strain, andmay take any type of dosage form. For example, the cosmetics prepared byusing the said cosmetic composition may include creams, packs, lotions,essences, toners, foundations and makeup bases, and may becommercialized in any dosage forms listed above to achieve the purposeof the present invention, but not limited thereto. The ingredientscomprised in the cosmetic composition of the present invention includethose commonly used in cosmetic compositions, in addition to the aboveingredients, for example, conventional adjuvants such as antioxidants,stabilizers, solubilizers, vitamins, pigments and fragrances, andcarriers.

In another embodiment of the present invention, the present inventionrelates to a functional patch comprising at least one selected from thegroup consisting of the cellular component of Lactobacillus gasseriKBL697 strain, cultures of said strain, lysates of said strain, andextracts of said strain, and said functional patch may be used forcosmetic or medical purpose.

The patch may be characterized by its function of alleviating at leastone sensitive skin condition selected from the group consisting ofcutaneous allergy, skin urticaria, atopic dermatitis, psoriasis, mycoticinfections and eczema, but not limited thereto.

In the present invention, the patch is typically a small adhesivebandage containing the substances to be delivered, and the bandage cantake a variety of forms. The simplest form is an adhesive single bodycomprising a reservoir containing the substances to be delivered placedon a support. The reservoir is typically formed from a cosmetically orpharmaceutically acceptable pressure sensitive adhesive, but in somecases may also be formed from non-adhesive materials provided with athin adhesive layer suitable for the skin contacting surface. The rateat which the substances to be delivered are administered from the patchto the subject wearing the patch can be changed because the permeabilityof the skin to the substances to be delivered usually depends onindividuals and the location of the skin, which can be easily selectedby those skilled in the art.

Hereinafter, the present invention will be described in more detailthrough examples. These examples are only for illustrating the presentinvention, and it will be apparent to those skilled in the art that thescope of the present invention is not to be construed as being limitedby these examples.

[16] Example 1. Alleviation and Therapeutic Effects by KBL697 forAllergic Reactions Resulting from Inhibition of Th2 Type Cytokines in TCells

Type-2 helper T cell (Th2)-related cytokines, such as IL-4 and IL-5,have been reported to contribute to chronic allergic reactions byincreasing Th2-related immune reactions and increasing IgE production(Passante E, Inflamm. Res. 2009). The present invention, by verifyingthe effect of inhibiting IL-4 and IL-5 secretion by variousLactobacillus strains, attempted to screen probiotic strains that can beused as therapeutic agents for various allergic diseases includingchronic allergic diseases. To this end, the effect of inhibiting thesecretion of IL-4 and IL-5 was tested as follows, using EL4 cell linewhich is the mouse T cell line.

[17] 1-1. Incubation of EL-4 Cell Lines

EL4 (ATCC NO. TIB-39) cells were cultured in DMEM medium supplementedwith 10% FBS (fetal bovine serum), penicillin (100 μg/mL), andstreptomycin (100 μg/mL) at 37° C. under 5% CO₂, and then subculturedonce every three days. EL4 cells were seeded onto a 24-well plate with aconcentration of 4×10⁵ cells/well, then cultured for 16 to 20 hours toinduce allergic reactions, and treated with strains.

[18] 1-2 Incubation of Strains and Recovery

The Lactobacillus gasseri strains to be used were cultured in MRS mediumsupplemented with 0.5% cysteine, were activated through a total of twosubcultures at 18-hour intervals, and then were used in experiments. Theresulting culture solution was centrifuged at 15,000×g for 3 minutes,and the pellet was washed with a PBS buffer. The strains were stainedfor 15 minutes with SYTO9 and PI by using LIVE/DEAD™ BacLight™ BacterialViability and Counting Kit for flow cytometry (Thermo Fisher Scientific,USA). Then, the contained beads were added, and the number of stainedlive bacteria was calculated by using a flow cytometry assay.

1-3. Strain Treatment and Measurement of an Amount of IL-4 and IL-5Secretion after Inducing Allergic Reactions

In order to induce allergic reactions in the EL4 cells previously seededonto a 24 well plate, each well was treated with 100 μL of PMA (20ng/mL) and ionomycin (1 μg/mL). Then, 300 μL of the previously preparedstrains was treated in the ratio of cell to strain of 1:10. Afterincubation for 24 hours in the 5% CO₂ incubator at 37° C., thesupernatant was collected, and Mouse IL-4 ELISA set (Cat NO. 555232, BDOptEIA™) and Mouse IL-5 ELISA set (Cat NO. 555236, BD OptEIA™) were usedto measure the amount of IL-4 and IL-5 secreted, according to themanufacturer's method.

As a result, as shown in FIGS. 1 and 2, it was confirmed that KBL697effectively inhibited the secretion of both IL-4 and IL-5, andtherapeutic and preventive effects thereof on allergies could beprovided through inhibition of secretion of Th2 type cytokine thatmediates an allergic reaction.

Example 2. Alleviation and Treatment Effects by KBL697 for AllergicReactions Resulting from Inhibition of Histamine Secretion in Basophils

It has been reported that, in an allergic reaction, histamine isexpressed in tissues, causing an inflammatory reaction, and suppressinghistamine secretion leads to alleviation of allergic symptoms byblocking the reaction by histamine. In the present invention, thealleviation of allergic reactions and the therapeutic effect thereonthrough inhibition of histamine secretion by KBL697 were furtherconfirmed. Further, in order to confirm whether the effect for treatingand preventing chronic allergies identified in Example 1 is an inherentattribute of Lactobacillus gasseri, or is a remarkable effectparticularly in KBL697 among Lactobacillus gasseri, the ability tosuppress histamine secretion was evaluated also for nine additionalLactobacillus gasseri strains in addition to KBL697. After inducingdegranulation after culturing the RBL-2H3 cell line, the ability tosuppress histamine secretion was tested by measuring the activity ofβ-hexosaminidase co-secreted with histamine using a colorimetricreaction with a substrate.

2-1. Incubation of RBL-2113 Cell Lines

RBL-2H3 (ATCC NO. CRL-2256) cells were cultured in DMEM mediumsupplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin (100μg/mL) at 37° C. under 5% CO₂, and then subcultured once every threedays. RBL-2H3 cells were seeded onto a 6-well plate with a concentrationof 1×10⁶ cells/well, then cultured for 3 hours, and then treated withIgE (0.5 μg/mL) to incubate for 16 to 20 hours.

2-2. Incubation of Strains and Preparation of Culture Solution

The Lactobacillus gasseri strains to be used were cultured in MRS mediumsupplemented with 0.5% cysteine, were activated through a total of twosubcultures at 18-hour intervals, and then were used in experiments. Theresulting culture solution was centrifuged at 15,000×g for 3 minutes tocollect the supernatant.

2-3. Inducing Degranulation after Treatment of Strains

After removing the medium of RBL-2H3 cells which were previously seededonto a 6-well plate, the cells were washed twice with 1 mL of Siraganianbuffer (pH 7.2). Thereafter, the cells were treated with 120 μL of thepreviously prepared bacterial culture solution or the positive controlgroup ketotifen (20 μg/mL) per well, incubated for 20 minutes in the 5%CO₂ incubator at 37° C., followed by treating with 60 μL of antigen(DNP-HAS, 1 μg/mL) to induce degranulation by the antigen-antibodyreaction. As a negative control, degranulation was induced aftertreating with 120 μL of the Siraganian buffer. The reaction was carriedout in the 5% CO₂ incubator at 37° C. for 20 minutes, and then thesupernatant was collected.

2-4. Identification of Color Reaction

To identify the activity of β-hexosaminidase, 25 μL of the supernatantcollected in Example 2-3 was transferred to each well of a 96-wellplate, and then 25 μL of substrate (p-nitrophenylN-acetyl-D-glucosaminidase) was added to each, and followed by areaction at 37° C. in the 5% CO₂ incubator for 90 minutes. Then, 200 μLof stop solution (Na₂CO₃/NaHCO₃) was added to stop the reaction and thenabsorbance was measured at 405 nm. The absorbance when treated with eachtype of Lactobacillus gasseri was compared to the negative control groupand converted to percentage to show the level of inhibition of thedegranulation.

As a result, as can be seen in FIG. 3 and Table 1, KBL697 showed up to 4times lower amount of β-hexosaminidase secretion compared to otherLactobacillus gasseri strains, which was confirmed to be lower than thatof the commercially available antihistamine agent ketotifen, a positivecontrol. As a result, it was found that KBL697 exhibited excellentdegranulation-preventing activity to effectively alleviate allergicsymptoms caused by excessive secretion of histamine.

TABLE 1 Strain under Treatment or β-hexosaminidase Species TreatmentDrug (fold change) - (Negative control) Siraganian buffer 1.0 -(Positive control) Ketotifen 0.3861 Lactobacillus gasseri KBL697 0.2213Lactobacillus gasseri KBL695 0.4601 Lactobacillus gasseri KBL698 0.4620Lactobacillus gasseri KBL685 0.4450 Lactobacillus gasseri KBL686 0.4213Lactobacillus gasseri KBL688 0.4571 Lactobacillus gasseri KBL373 0.5691Lactobacillus gasseri SNUG50243 0.5309 Lactobacillus gasseri SNUG600801.0091 Lactobacillus gasseri SNUG60134 0.9236

Example 3. Analysis of Immunoregulatory and Inflammation InhibitoryEffects of KBL697

Immunoregulatory and inflammatory inhibitory effects of KBL697 were alsoverified in addition to the anti-allergic efficacy thereof. To this end,the ratio between IL-10, a representative cytokine that has animmunomodulatory function, and TNF-α and IL-6, which are cytokines asthe major indicators of an inflammatory reaction (IL-10/TNF-α,IL-10/IL-6) was measured by using macrophage, which plays a criticalrole in the inflammatory reaction.

3-1. Incubation of RAW264.7 Cell Lines

RAW264.7 (ATCC NO. TIB-71) cells were cultured in DMEM mediumsupplemented with 10% FBS, penicillin (100 m/mL), and streptomycin (100m/mL) at 37° C. under 5% CO₂, and then subcultured once every threedays. RAW264.7 cells as incubated were seeded onto a 24-well plate withthe concentration of 1×10⁵ cells/well, then cultured for 16 to 20 hours,and then used for Example 3-3.

3-2. Incubation of Strains and Preparation of Samples

The culture solutions were corrected with the same number of livebacteria and then prepared as three samples: live bacteria,pasteurization and heat killing. The samples for live bacteria,pasteurization and heat killing were prepared according to the samemethod as that of Example 1-2. Then, the sample for pasteurization wasreacted at 70° C. for 30 minutes and prepared through the samecentrifugation process. The sample of heat killing was sterilized at121° C. for 15 minutes and prepared through the same process. As anegative control group, the MRS medium supplemented with 0.5% cysteinewas used.

3-3. Strain Treatment and Measurement of an Amount of InflammatoryCytokines Secreted After Inducing Inflammatory Reactions

In order to induce inflammatory reactions in RAW264.7 cells previouslyseeded onto a 24-well plate, each well was treated with 500 μL of LPS(20 ng/mL). Then, 300 μL of the previously prepared strains was treatedin the ratio of cell to strain of 1:10, and incubated for 24 hours inthe 5% CO₂ incubator at 37° C. The supernatant in the culturedcell-strain mixture was collected and Mouse TNF ELISA Set II (Cat No.558534, BD OptEIA™), Mouse IL-6 ELISA Set (Cat No. 555240, BD OptEIA™),and Mouse IL-10 ELISA Set (Cat No. 555252, BD OptEIA™) were used tomeasure an amount of each cytokine, according to the manufacturer'smethod.

As a result, as shown in FIG. 4, it was confirmed that especiallyKBL697, among Lactobacillus gasseri strains, induced IL-10 secretion andthus showed excellent effects in terms of immunoregulation andinflammatory reaction suppression. As shown in FIGS. 5 and 6, from theability to secrete IL-10 corrected with the pro-inflammatory cytokinesTNF-α and IL-6, it was confirmed that the KBL697 treatment group showedthe inflammation inhibitory effect and immunoregulatory ability muchenhanced compared to the control group. Accordingly, it has been foundthat KBL697 has immunoregulatory and inflammation inhibitory activitiesthrough IL-10 secretion. In addition, these effects were similarlyconfirmed not only in live bacteria but also in pasteurized or heatkilled dead bodies (see Table 2 and Table 3), indicating that KBL697 canbe used to suppress inflammatory reactions in various forms such as deadbacteria.

TABLE 2 IL-10/TNF-a Live Heat-killed Pasteurized Control −2.4509342−2.0474707 −1.9242497 (N.D.) (N.D.) (N.D.) KBL697 1.81114395 6.281303884.43515847 N.D.: Not Determined

TABLE 3 IL-10/IL-6 Live Heat-killed Pasteurized Control −6.010433−4.6669667 −4.5960301 (N.D.) (N.D.) (N.D.) KBL897 2.4709538 8.16652685.65439003 N.D.: Not Determined

Example 4. Analysis of Anti-Fungal Effects of KBL697

The anti-fungal effect of KBL697 was confirmed by a spot assay method.About 1% of KBL697 was inoculated on MRS liquid medium, and then wasincubated in the 37° C. incubator for about 24 hours under anaerobicconditions for stationary culturing. The culture solution in which thecells were incubated was spotted in the MRS solid medium, which wasprepared under anaerobic conditions, in an amount of 10 μL at each time,and then incubated at 37° C. under anaerobic conditions for about 24hours. Malassezia furfur KCTC 7545, a fungal microorganism, was preparedby inoculating it in the mYPG liquid medium, which was prepared underaerobic conditions, at a rate of 1% and followed by incubation in the37° C. incubator for about 24 to 48 hours. The mYPG soft medium to beused for anti-fungal efficacy evaluation was prepared with thecomponents shown in Table 4, and 2.5 mL of the prepared culture mediumwas inoculated with the culture solution in which 500 μL of M. furfurwas incubated. 2.5 mL of mYPG soft medium inoculated with M. furfur waspoured into the MRS medium spotted with KBL697 and dried for 1 hour. Thedried medium was incubated under aerobic conditions in the 37° C.incubator for 24 to 48 hours. When a clear zone was identified in thecultured medium, anti-fungal activity was determined by measuring thelength from the outside of the spotted lactic acid bacteria to the clearzone. The clear zone is a part where growth of fungi was inhibited, andthe anti-fungal activity caused by lactic acid bacteria was determinedthrough the length to the clear zone.

As a result of three repeated experiments, the clear zones wereidentified at intervals of about 6.00 mm at the part spotted with KBL697(FIG. 7), which indicates that KBL697 can effectively suppress thegrowth of fungal microorganisms.

TABLE 4 Components Proportion (g/l) Malt extract 5.0 Peptone 10.0Glucose 20.0 Tween 40 1.0 Tween 80 1.0 Agar 4.5

Example 5. Effects of KBL697 on Alleviation of Atopic Conditions

In order to verify the effect on atopic alleviation among the allergyimprovement effects of KBL697, the NC/Nga mouse model, an animal modelof atopic skin disease, was used.

After dividing NC/Nga mice into groups of five mice, the back of eachmouse was epilated from the lower ear to the upper tail and mice wereleft for 24 hours. Then, 200 μL of a 1% DNCB (dinitrochlorobenzene)solution (acetone:olive oil=3:1) was applied twice a week onto theepilated portion to induce atopic dermatitis. From the third week ofdermatitis induction, 200 μL of PBS was orally administered to the micein the control group daily; the cultured KBL697 strain was centrifuged,washed through dilution with PBS and recovery, and then prepared so thatat least 2×10⁹ (KBL697-9)/2×10⁸ (KBL697-8)/2×10⁷ (KBL697-7) CFU could beadded to 200 μL of PBS, which was orally administered to the mice in thetest group in 200 μL/day. Meanwhile, 200 μL of dexamethasone (60 μg/mL)was administered to the mice in the positive control group. Then, duringthree weeks of administration of the bacteria, dermatitis scores of themice in the control and test groups were measured weekly, and on the 3rdweek after the administration of the bacteria, the mouse's scratchingtime and skin thickness, and IgE concentration-in-blood after conductingautopsies of mice were measured.

5-1. Evaluation of Dermatitis Score

To evaluate DNCB-induced skin lesions, the dermatitis score was measuredthrough the following method. Skin conditions were monitored by takingpictures for 3 weeks at one week intervals from the 3rd week since thestrain was administered. Four indicators of dryness, edema,erythema/hemorrhage, and erosion/excoriation of the skin were checked.And a condition with no lesions was scored as point 0, a mild conditionas point 1, a moderate condition as point 2, and a severe condition aspoint 3, and the total score was evaluated.

As a result, as shown in FIG. 8, the dermatitis score induced by DNCBwas significantly reduced in the group dosed with KBL697, compared tothe control group (negative) where atopic dermatitis was induced, and inparticular, the test group (KBL697-9) that KBL697 was administered at2×10⁹ CFU showed the dermatitis score even lower than the positivecontrol group of dexamethasone administration. As a result, the effectof treating atopic dermatitis according to the administration of KBL697was verified.

5-2 Itching Relief Effects

In order to verify the effect of alleviating itching according to theadministration of KBL697 in the mouse models suffering from atopicdermatitis induced by DNCB, the scratching time was measured by taking avideo of the mouse models for 10 minutes after 3 weeks of strainadministration.

As a result, as can be seen in FIG. 9, it appeared that the scratchingtime was significantly reduced in all test groups that KBL697 wasadministered, compared to the PBS administration group, which confirmedthat the itching symptoms of atopic dermatitis were much alleviated bythe administration of KBL697.

5-3. Decrease of Skin Thickness

In order to verify the effect of alleviating itching afteradministration of KBL697 to mouse models suffering from atopicdermatitis induced by DNCB, the mouse ear thickness and dorsal skinthickness were measured with calipers three weeks after the strain wasadministered, and the relief of edema symptom due to atopic dermatitiswas observed.

As a result, as can be seen in FIGS. 10 and 11, it was observed that inthe test group dosed with KBL697 and the positive control group, the earand dorsal skin thicknesses were significantly reduced.

5-4. Decrease in IgE Concentration-in-Blood

It has been found that the concentration of IgE in patients havingatopic dermatitis has mostly increased as clinical severity of atopicdermatitis increased (Matsumoto M, J. Immunol. 1999). Thus, theconcentration of IgE, a representative hematologic factor appearing asatopic dermatitis arises, was measured by collecting blood three weeksafter the strain was administered, separating serum therefrom and usingMouse IgE ELISA Set (Cat No. 555248, BD OptEIA™).

As a result, as shown in FIG. 12, it was found that the concentration ofIgE-in-blood was significantly decreased in the test group that KBL697was orally administered, which indicated the anti-allergic effect afteradministration of KBL697.

Example 6. Effects of KBL697 on Strengthening Mesenteric Tight Junction

In the case of colitis patients, the expression of proteins involved inthe tight junction of enterocytes decreases, resulting in higher cellpermeability, which leads to more severe inflammatory reactions (J.Landy, World J. Gastroenterol., 2016). In this regard, the presentinvention attempted to confirm whether KBL697 could enhance tightjunction in Caco-2 cell lines, a representative intestinal epithelialcell model.

6-1. Incubation of Caco-2 Cell Lines

Caco-2 (ATCC NO. HTB-37) cells were incubated in the MEM mediumsupplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin (100μg/mL) at 37° C. under 5% CO₂, while replacing the medium once every 2days, and then subcultured once every 4 days.

6-2. Measurement of Tight Junction

200 μL of Caco-2 cells was seeded onto the upper layer portion of the 24Transwell plate (Corning, USA) at a concentration of 3×10⁵ cells/mL, andthen incubated for seven days. On the next day, after replacing themedium with that having only MEM without FBS, the cells were incubatedovernight, and a chopstick electrode set was inserted thereto to measureTEER (transepithelial electrical resistance) by using EVOM EpithelialTissue Voltohmmeter (World Precision Instruments, Florida, USA) (0 hTEER). Then, the strains were treated so that the ratio of cell tostrain would be 1:100, and incubated for 24 hours, and then TEER wasmeasured (24 h TEER).

After calculating by the equation:

TEER (Ω·cm²)=(resistance (Ω)−background resistance (Ω))×membrane area(cm²), the following equation applied:

TEER change (%)=24 h TEER (Ω·cm²)/0h TEER (Ω·cm²)×100

As a result, as shown in FIG. 13, the Caco-2 cell lines treated withKBL697 showed that the tight junction increased by 20%, compared to thecontrol group, indicating that KBL697 has an effect of restoring theweakened intercellular junctions between enterocytes due to colitis. Onthe other hand, such an effect was not found in other Lactobacillusgasseri strains KBL342 and KBL381.

Example 7. Effects of KBL697 on Treatment and Prevention of Colitis

In order to verify the therapeutic effect of KBL697 on colitis, mousemodels in which colitis was induced were used. The mouse models weredivided into groups of five C57BL/6 mice, and then fed tap water with 3%DSS dissolved therein for 5 days, and thereby inducing colitis.Subsequently, the mice in the positive control group were orallyadministered with the steroid-based drug prednisolone in 200 μL of PBSin accordance with 1 mg/kg/day, and the mice in the test group weredaily provided via an oral administration with 200 μL of KBL697 strainswhich were prepared in a same manner as in Example 5 and then eachdiluted to be 5×10⁹ CFU/mL, 5×10⁸ CFU/mL, and 5×10⁷ CFU/mL. The mice inthe control group were orally administered with 200 μL of PBS daily. ThePBS used in this example was prepared so that 0.05% of cysteine could beadded to rule out the effect of small amounts of cysteine which wasincluded in the composition of the strain culture solution and remainedeven after washing, according to the report that cysteine was associatedwith anti-inflammation in some cells (Hasegawa, S et al., Clin ExpImmunol. 2012, 167, 269-27). From the day the DSS began to be consumeduntil the 21^(st) day, the weight changes of mice in the control andtest groups were measured daily, and on the 21^(st) day after the DSSwas supplied, the mice were subjected autopsies to measure the length ofthe colon.

As a result, as shown in FIG. 14, it was found that in thecolitis-induced mice dosed with 1×10⁹ CFU of KBL697, similarly to themice in the control group, the body weight was decreased rapidly until10^(th) day, and then gradually recovered to restore normal levels onthe 21^(st) day. In particular, as shown in FIG. 15, in the mice orallyadministered with 1×10⁹ CFU of KBL697 (KBL697×10{circumflex over ( )}9),it was found that the length of the colon was recovered to 90% level ofthe Naive group, and the symptom of reducing the length of the colon wassignificantly alleviated, compared to the negative control group of lessthan 80%. As a result, it was found that KBL697 showed an effect ofimproving colitis disease.

Example 8. Effects of KBL697 on Treatment and Prevention of Psoriasis

In order to verify the effects of KBL697 on alleviation of psoriasis,Balb/c mouse models were used.

After dividing Balb/c mice into groups of five mice, the mice in thecontrol group were orally administered with 200 μL of PBS daily for tendays, while the mice in the test group were orally administered with 200μL of each test strain diluted in PBS at least 1×10⁹ CFU/0.2 mL daily.Strains and the control group were prepared in a same manner as Example7. Two weeks after administering the bacteria, the back of each mousewas epilated from the lower end of the ear to the middle of the back,and mice were left for 24 hours. Then, 62.5 mg of 5% imiquimod cream wasapplied to the epilated portion once a day, causing psoriasis. Afterinducing psoriasis, the mice in the positive control group wereadministered with 200 μL of dexamethasone (0.25 mg/mL) daily. The sameamount of KBL697 was administered daily even during psoriasis wasinduced. For ten days that psoriasis was induced, psoriasis area andseverity index (PAST) of mice in the control and test groups wasmeasured at two day intervals, and on the 10th day after psoriasis wasinduced, the skin thickness of each mouse was measured, and then themouse was subjected to autopsy to determine the concentration ofcytokines in skin tissues and in blood.

8-1. PASI Measurement and Changes in Skin Thickness

In order to evaluate imiquimod-induced skin psoriasis lesions, the PASIscore was measured by the following method. Three indicators oferythema, scaling, and thickness of the skin were checked. And acondition with no lesions was scored as point 0, a mild condition aspoint 1, a moderate condition as point 2, a severe condition as point 3,and a very severe condition as point 4, and the total score wasevaluated.

As a result, as shown in FIGS. 16A-E, PASI was significantly reduced inthe imiquimod-induced psoriasis group dosed with KBL697 similarly to thepositive control group dosed with dexamethasone (DXM), compared to thecontrol group (NC) where psoriasis was induced. In addition, as a resultof measuring the ear and dorsal skin thicknesses of mice using calipersand observing the effect on relief of edema symptom due to psoriasis, asshown in FIGS. 16F and 16G, it was found that the ear and dorsal skinthicknesses were significantly reduced in the test group dosed withKBL697, similarly to the positive control group.

8-2. Cytokine Measurement

In the case of psoriasis, reactions of Th1 cytokines such as IFN-γ andTNF-α, as well as Th17 responses, representatively IL-17, have beenknown to play an important role in the development and exacerbation ofsymptoms (Brembilla N C, Front Immunol. 2018). Mice with psoriasisinduced by imiquimod were subjected to autopsies to determine theconcentration of cytokines in skin tissues and in blood. Theconcentration of cytokines was determined with the protein sample takenin skin tissues and the serum sample prepared from blood in a samemanner as Example 5-4 by using each of Mouse TNF-α ELISA Set (Cat No.555138, BD OptEIA™) Mouse IFN-γ ELISA Set (Cat No. 558534, BD OptEIA™),Mouse IgE ELISA Set (Cat No. 555248, BD OptEIA™), and Mouse IL-17 DuoSetELISA (Cat No. DY421-05, R&D SYSTEMS) kit.

As can be seen in FIGS. 17A-D, it was confirmed that inflammatorycytokines such as TNF-α and IFN-γ in skin tissues increased inpsoriasis-induced mice were reduced by KBL697. In addition, it wasconfirmed that IL-17 specific to Th17 was also increased inpsoriasis-induced skin tissues and serum, but decreased by KBL697administration. As a result, it was confirmed that the administration ofKBL697 could reduce the inflammatory cytokines which increasedspecifically when psoriasis was onset, exhibiting effects on thetreatment or prevention of the disease through the reduction effect.

Example 9. Effects on Treatment and Prevention of Colitis UponConcurrent Administration of Antibody for Treating Colitis and KBL697

Infliximab (product name Remicade) is a therapeutic recombinant antibodydrug used as an injection for autoimmune diseases such as rheumatoidarthritis, ankylosing spondylitis, ulcerative colitis, Crohn's diseasein adults, Crohn's disease in children, psoriasis, and psoriaticarthritis. The purpose of this study was to confirm the effect ofimproving intestinal disease symptoms in vivo in the case of concurrentadministration of KBL697 and infliximab.

After dividing C57BL/6 mice into groups of eight mice, the mice were fedtap water with 2% DSS dissolved therein for 8 days (DO to D7), inducingcolitis. At the same time, 200 μL of PBS was orally administered to themice in the control group for 14 days (D0 to D13) daily, while 200 μL ofthe KBL697 strain diluted in PBS to 1×10¹⁰ CFU/mL was orallyadministered to the mice in the group dosed with KBL697 so that theamount of daily administration could be set at 2×10⁹ CFU. In thetherapeutic antibody administration group, infliximab antibody wasadministered once every 3 days to be a dose of 5 mg/kg per mouse, and200 μL of PBS was orally administered daily during the period that theKBL697 strain was administered. In the test group for concurrentadministration, 200 μL of the solution of KBL697 strain diluted in PBSto be 1×10¹⁰ CFU/ml was orally administered every day, and on the thirdday, infliximab antibody was injected intravenously to be a dose of 5mg/kg per mouse. The test groups without infliximab antibodyadministration were injected intravenously with the same volume of PBS.During the 14 days in which colitis was induced by DSS, the body weightchanges of the mice in the control group and each test group weremeasured daily, and on the 14^(th) day after DSS was supplied (D14),mice were subjected to autopsies to measure the length of the colon.

As a result, as shown in FIGS. 18A and 18B, regarding the colon lengthchange, the width of decrease in the colon length was significantlyimproved in the groups that KBL697 was administered alone (DSS+KBL697)and that infliximab was administered alone (DSS+IFX), compared to themice in the control group (DSS+PBS). In the group that infliximab andKBL697 were concurrently administered (DSS+Combine), it was confirmedthat there was a significant improvement in colon length compared to thegroup that infliximab or KBL697 was administered alone.

Regarding the change in body weights, as shown in FIGS. 18C and 18D,when 14 days elapsed after the administration, the effect on body weightdecrease was significantly improved in the groups that KBL697 wasadministered alone (DSS+KBL697) and that infliximab was administeredalone (DSS+IFX), compared to the mice in the control group with notreatment. When three days elapsed after the administration, it wasconfirmed that high body weights were maintained in the group thatinfliximab and KBL697 were concurrently administered (DSS+Combine),compared to all test groups that DSS was administered, verifying theeffect of alleviating the symptom of body weight decrease due toinducement of colitis. Accordingly, regarding the use of said strain forthe treatment of irritable bowel syndrome, it was confirmed that astronger therapeutic effect could be expected when the strain wasadministered in combination with the commercially available therapeuticantibodies.

Specific aspects of the present invention have been described in detailabove, and it is obvious to those skilled in the art that these specificaspects are only preferred embodiments, and the scope of the presentinvention is not limited thereby. Therefore, the scope of the presentinvention is substantially defined by the following claims, withequivalents to the claims

Name of Depository Organization: Korean Collection for Type Cultures,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181,Ipsin-gil, Jeongeup-si, Jeolllabuk-do, 56212, Republic of Korea.

Accession No.: KCTC13520BP

Accession Date: 20180427

INDUSTRIAL APPLICABILITY

The strain of Lactobacillus gasseri KBL697 (Accession No. KCTC 13520BP)according to the present invention attenuates allergic reactions ofcells, significantly improves symptoms of atopic dermatitis, andexhibits anti-inflammatory, immunoregulatory and anti-fungal effects anda therapeutic effect on intestinal diseases such as irritable bowelsyndrome and colitis. Thus, the single strain alone can achieve all thepurposes of alleviating allergic diseases and inflammatory diseases,improving intestinal health, and immunoregulation, thereby findingadvantageous applications as a probiotic substance. In addition, thestrain, based on the anti-fungal activity thereof, can be advantageouslyutilized in a skin external preparation against various skin diseasescaused by fungi, and in a cosmetic composition and a functional patchfor alleviating sensitive skin.

SEQUENCE LIST FREE TEXT

An electronic file of Sequence List attached

1. A strain of Lactobacillus gasseri KBL697 with Accession No. KCTC13520BP.
 2. The strain according to claim 1, wherein said straincomprises 16S rDNA sequence of SEQ ID NO:
 1. 3. A food compositioncomprising at least one selected from the strain according to claim 1,cultures of said strain, lysates of said strain, and extracts of saidstrain.
 4. The food composition according to claim 3, wherein saidcomposition is a health functional food composition having at least oneeffect selected from alleviation of allergic symptoms, alleviation ofinflammatory symptoms, improvement of intestinal health, andimmunoregulation.
 5. The food composition according to claim 4, whereinsaid allergic symptoms are selected from atopic dermatitis, eczema,allergic asthma, allergic rhinitis, allergic conjunctivitis or foodallergy; said alleviation of intestinal health is the alleviation of atleast one selected from abdominal bloating, abdominal discomfort,infectious diarrhea caused by pathogenic microorganisms, gastrocolitis,inflammatory bowel diseases, neurogenical intestinitis syndrome,irritable bowel syndrome, overgrowth of small intestinal microorganismsand intestinal feeding diarrhea.
 6. A feed composition comprising atleast one selected from the strain according to claim 1, cultures ofsaid strain, lysates of said strain, and extracts of said strain.
 7. Ananti-fungal composition comprising at least one selected from the strainaccording to claim 1, cultures of said strain, lysates of said strain,and extracts of said strain.
 8. The anti-fungal composition according toclaim 7, wherein said composition exhibits anti-fungal activities toMalassezia furfur.
 9. A skin external preparation comprising theanti-fungal composition according to claim
 7. 10. A medical patch forimproving cutaneous allergy, skin urticaria, atopic dermatitis,psoriasis, mycotic infection or eczema, comprising at least one selectedfrom the strain according to claim 1, cultures of said strain, lysatesof said strain, and extracts of said strain.
 11. A pharmaceuticalcomposition for the treatment or prevention of allergic disease,inflammatory disease, intestinal disease or autoimmune disease,comprising at least one selected from the strain according to claim 1,cultures of said strain, lysates of said strain, and extracts of saidstrain.
 12. The pharmaceutical composition according to claim 11,wherein said allergic disease is selected from eczema, allergic asthma,allergic rhinitis, atopic dermatitis, allergic conjunctivitis or foodallergy.
 13. The pharmaceutical composition according to claim 11,wherein said inflammatory disease is selected from edema,conjunctivitis, periodontitis, rhinitis, otitis media, chronicsinusitis, pharyngolaryngitis, tonsillitis, bronchitis, pneumonia,gastric ulcer, gastritis, colitis, gout, eczema, acne, atopicdermatitis, contact dermatitis, seborrheic dermatitis, ankylosingspondylitis, rheumatic fever, fibromyalgia, osteoarthritis, psoriaticarthritis, rheumatoid arthritis, peri-arthritis of the shoulder,tendinitis, tenosynovitis myositis, hepatitis, cystitis, nephritis,Sjogren's syndrome, myasthenia gravis, sepsis, vasculitis, bursitis,temporal arteritis or multiple sclerosis.
 14. The pharmaceuticalcomposition according to claim 11, wherein said intestinal disease isselected from abdominal bloating, abdominal discomfort, infectiousdiarrhea caused by pathogenic microorganisms, gastrocolitis,inflammatory bowel diseases, neurogenical intestinitis syndrome,irritable bowel syndrome, overgrowth of small intestinal microorganismsor intestinal feeding diarrhea.
 15. The pharmaceutical compositionaccording to claim 11, wherein said autoimmune disease is selected frompsoriasis, psoriatic arthritis, rheumatoid arthritis, lupus, systemicscleroderma, atopic dermatitis, asthma, Guilian-Barre syndrome,myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis,autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto'sthyroiditis, multiple sclerosis, temporal arteritis, juvenile diabetes,alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolyticanemia, Wegener's granulomatosis, Sjogren's syndrome, Addison's disease,Crohn's disease, or Behcet's disease.
 16. The pharmaceutical compositionaccording to claim 11, wherein said pharmaceutical composition is usedin combination with at least one drug selected from infliximab,adalimumab, golimumab, abciximab, alemtuzumab, atlizumab, basiliximab,belimumab, bevacizumab, ipilimumab, brentuximab vecotin, canakinumab,capromab pendetide, catumaxomab, certolizumab pegol, cetuximab,daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, efungumab,ertumaxomab, etanercept, etaracizumab, fontolizumab, gemtuzumabozogamicin, girentuximab, ibritumomab tiuxetan, igovomab, imciromab,ipilimumab, labetuzumab, mepolizumab, motavizumab, natalizumab,nimotuzumab, nofetumomab merpentan, ofatumumab, omalizumab, oregovomab,palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab,raxibacumab, rituximab, rovelizumab, ruplizumab, tacatuzumab tetraxetan,tefibazumab, tocilizumab, tositumomab, trastuzumab, ustekinumab,secukinumab, vedolizumab, visilizumab, votumumab, zalutumumab andzanolimumab.
 17. A method for treating allergic disease, inflammatorydisease, intestinal disease or autoimmune disease, comprisingadministering at least one selected from the strain according to claim1, cultures of said strain, lysates of said strain, and extracts of saidstrain to a subject in need thereof.
 18. Use of a composition forpreparing a preventive or therapeutic drug for allergic disease,inflammatory disease, intestinal disease or autoimmune disease,comprising at least one selected from the strain according to claim 1,cultures of said strain, lysates of said strain, and extracts of saidstrain.
 19. A cosmetic composition comprising at least one selected fromthe strain according to claim 1, cultures of said strain, lysates ofsaid strain, and extracts of said strain.
 20. The cosmetic compositionaccording to claim 19, wherein the cosmetic composition improvescutaneous allergy, skin urticaria, atopic dermatitis, psoriasis, mycoticinfection or eczema.
 21. The cosmetic composition according to claim 19,wherein the cosmetic composition is a cosmetic patch.